The invention relates to a method for preparing polypeptides, and in particular virus-like particles (VLPs) or capsomers of human papillomavirus.
The papillomaviruses are a group of small DNA viruses, which induce warts and other lesions in a variety of higher vertebrates including humans.
Papillomaviruses (PV) are members of the genus Papillomavirus, family Papillomaviridae, and contain a double stranded circular DNA genome with a typical size of 7 900 base pairs (Seedorf et al., 1985). All PVs have a similar genomic organisation, with an early gene region encoding proteins involved in DNA replication and cellular transformation, and a late region encoding the viral capsid proteins (FIG. 1). A non-coding region known as the long control region (LCR) contains control elements for transcription and replication.
Papillomaviruses encode two viral structural proteins, L1 and L2. The virion contains 360 L1 molecules arranged as 72 capsomers, each of which is a pentamer composed of five L1 molecules (Baker et al., 1991). The ratio of L1 to L2 molecules has been estimated as approximately 30:1 (Doobar et al., 1987), which suggests that each virion would contain approximately twelve L2 molecules. The greater number of L1 molecules per virion has led to L1 being referred to as the ‘major’ capsid protein and L2 being referred to as the ‘minor’ capsid protein.
HPV-16 L1 is encoded by a 1.518 kb gene, giving rise to a protein of 504 amino acids. L1 has a molecular weight of 55 to 58 kD (Browne et al., 1988). Domains of L1 are likely to mediate cell binding and to contain antigenic determinants mediating antibody and T cell immune responses to the virus.
Among the genital human papillomaviruses (HPVs), there are low risk HPVs (for example, HPV 6 and HPV 11) that cause genital warts and cervical lesions that usually regress or do not progress to malignancy, and high risk (or oncogenic) genotypes (for example, HPV 16 and HPV 18), which are associated with high-grade cervical lesions and carcinomas. HPVs have also been implicated as the etiological agents in several other anogenital and upper aerodigestive tract cancers (Breitburd et al., 1999). A compelling body of clinical, molecular, experimental and epidemiological evidence has established that certain HPV types are the main cause of cervical cancer (Lowy et al., 1994; IARC, 1995).
HPV 16 is present in most cases of cervical cancer cases and an additional three types (HPV 18, 31 and 45) are present in approximately an additional 30% of cases (IARC, 1999).
Although the incidence of cervical cancer is decreasing in the US, it is the most common malignancy in women in developing countries, with about 500 000 new cases diagnosed each year.
Traditionally most prophylactic vaccines have consisted of live, attenuated virus or formalin inactivated virus. Papillomavirus virions are highly immunogenic, inducing high titres (>10 000) of neutralising antibodies when systemically inoculated (Doretzky et al., 1980; Kirnbauer et al., 1991, 1992). However, due to the difficulties and risks involved in generating large quantities of these traditional vaccines there has been great emphasis on the development of viral protein subunit or virus-like particle (VLP) vaccines.
The best candidate protein for a prophylactic vaccine against HPV is the major capsid protein L1, which self-assembles into VLPs (Schiller and Lowy, 2001). These VLPs are very well characterised, and morphologically appear indistinguishable from whole virions (Chen et al., 2001; Rose et al., 1993). Injection of VLPs into experimental animals induces neutralising antibodies (Rose et al., 1998); preliminary human trials of injected VLP vaccines have also shown that these are well tolerated and highly immunogenic, and in the former case, stimulated robust B and T cell responses (Evans et al., 2001; Harro et al., 2001).
An effective, cheap prophylactic vaccine against oncogenic types of mucotropic HPVs could potentially have an impact on the world cancer burden, especially against HPV 16.
A common-neutralizing epitope for HPV types 6 and 16 has been found in the region (aa) 108-120 of the HPV 16 minor capsid protein, L2 (Kawana et al., 1998, 1999). Balb/c mice that were nasally immunised with a synthetic peptide corresponding to the epitope elicited an immune response that resulted in IgA and IgG antibodies cross-reacting with L1/L2 capsids of HPV 6, 16 and 18 (Kawana et al., 2001). Immunisation of rabbits with either of two overlapping peptides derived from the L2 sequence region 94-122 from either Rabbit oral papillomavirus (ROPV) or Cottontail rabbit papillomavirus (CRPV) resulted in sera which reacted to purified cognate L2, specifically recognised L2 in infected cells, and neutralised virus in vitro. Rabbits immunised with CRPV peptides were immune to CRPV challenge (Embers et al., 2002).
The inventors therefore decided to further investigate the presentation of this L2 epitope on chimaeric L1 VLPs as a vaccine in its own right, and as a model for the presentation of other immunogenic peptide sequences.